The amplification of nucleic acids is commonly used in research, forensics, medicine, including diagnostics, and agriculture. One of the best-known amplification methods is the polymerase chain reaction (PCR), which is a target amplification method (See U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159). A PCR reaction typically utilizes two primers, which are bound to the 5′-end and 3′-end of the target nucleotide sequence and a DNA polymerase which extends the bound primers by adding bases using deoxynucleoside triphosphates (dNTPs) to generate double-stranded products. By raising and lowering the temperature of the reaction mixture, the two strands of the DNA product are separated and serve as templates for the next round of primer binding and extension, and the process is repeated. PCR requires a thermocycler instrumentation to raise and lower the temperature and thus has limitations in some rapid and field testing settings.
Target amplification methods in isothermal environments have been developed in the past few years. One is Strand Displacement Amplification (SDA). SDA combines the ability of a restriction endonuclease to nick the unmodified strand of a target DNA and the action of an exonuclease-deficient DNA polymerase to extend the 3′ end at the nick and displace the downstream DNA strand. The displaced strand serves as a template for a complementary strand reaction and vice versa, resulting in amplification of the target DNA (See U.S. Pat. Nos. 5,455,166 and 5,470,723). In the originally-designed SDA, the DNA was first cleaved by a restriction enzyme in order to generate an amplifiable target fragment with defined 5′ and 3′-ends but the requirement of a restriction enzyme cleavage site limited the choice of possible target DNA sequences (See for example, Walker et. al., Proc. Natl. Acad. Sci. USA 89:392-396 (1992)). SDA was further developed by the addition of bumper primers which flank the region to be amplified (Walker et al. supra (1992), U.S. Pat. No. 5,916,779). SDA technology has been used mainly for clinical diagnosis of infectious diseases such as chlamydia and gonorrhea. However, SDA is inefficient at rapidly amplifying sequences.
Another isothermal amplification system, Transcription-Mediated Amplification (TMA), uses the function of an RNA polymerase to make RNA from a promoter engineered in the primer region, and a reverse transcriptase, to produce DNA from the RNA templates. This RNA amplification technology has been further developed by introducing a third enzymatic activity, RNase H, to remove the RNA from cDNA without the heat-denaturing step. Thus the thermo-cycling step has been eliminated, generating an isothermal amplification method named Self-Sustained Sequence Replication (3SR) (See, for example, Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990)). However, the starting material for TMA and 3SR is limited to RNA molecules, and cannot be DNA.
A third isothermal target amplification method, Rolling Circle Amplification (RCA), generates multiple copies of a sequence for the use in in vitro DNA amplification adapted from in vivo rolling circle DNA replication (See Fire and Xu, Proc. Natl. Acad. Sci. USA 92:4641-4645 (1995); Lui, et al., J. Am. Chem. Soc. 118:1587-1594 (1996); Lizardi, et al., Nature Genetics 19:225-232 (1998), U.S. Pat. Nos. 5,714,320 and 6,235,502). A DNA polymerase extends a primer on a circular template generating tandemly linked copies of the complementary sequence of the template (See Kornberg and Baker, DNA Replication, W.H. Freeman and Company, New York (2nd ed. (1992)). Recently, RCA has been further developed in a technique, named Multiple Displacement Amplification (MDA), which generates a more uniform representation in whole genome amplification (See Dean et. al., Proc. Natl. Acad. Sci. USA 99:5261-5266 (2002)). However, these methods are inconvenient to use as there is a need to generate a circular template as part of the procedure.
A further isothermal amplification system, loop mediated isothermal amplification (LAMP), uses oligonucleotide primers provided at the 5′-side portion of each primer with a nucleotide sequence that is reverse complementary to a sequence of a region extended with this primer as the origin of extension (Notomi T, et al., 2000. Nucleic Acids Research 28:E63; and U.S. Pat. No. 6,410,278). Amplification proceeds in 45 min to 1 hour and yields a ladder pattern of various products. However, the need to extend the primers with the multiple target regions for both the forward and reverse directions of the template make primer design difficult. LAMP uses turn-back primers which includes a tail region at the 5′ end that folds back after the primer binds to the target sequence. Specifically, after the binding of the turn-back primer to the target sequence, the 5′ end tail of the turn-back primer will “fold back” and bind to a nucleotide sequence present on the target, thus forming a loop after the 3′ end of the turn-back primer binds to the target and is extended. The complementary strand will form another loop with a complementary sequence. Both loops are at least about 80 to 90 base pairs long. In addition, a turn-back primer construct must typically contain at least 80 base pairs of target sequence, excluding the 5′-side portions of the primers, and typically at least 200 base pairs of target sequence, including the 5′-side portions of the primers. Amplifying relatively large products restricts the reaction yield and lengthens reaction time.
Another isothermal amplification system is the Smart Amplification Process 2 (SMAP2) which utilizes a turn-back primer as described in LAMP, a folding primer, two outer primers and a booster primer. Examples are described in Mitani et al, Nature Methods, Vol. 4 No. 3: 257-262 (2007) and Kimura et al, Biochemical and Biophysical Research Communications 383 (2009): 455-459. The folding primer includes a palindromic sequence at the 5′ end that causes the formation of a small hairpin structure which is 3 to 15 base pairs long, with the hairpin loop part of the structure being 3-7 bases long.
The potential uses for nucleic acid amplification techniques continue to grow. For example, most nucleic acid assays, including many genotyping assays, utilize amplification reactions. Detection of environmental and food contaminants places demands on sensitivity and analytic power of diagnostic tests, which particularly need nucleic acid amplification procedures. Consequently, improvements in amplification methodology over current technologies are desirable. For example, desired improvements would include nucleic acid amplification methods which take place in isothermal reaction environments, involve convenient design of primers and other starting materials, and are capable of rapidly amplifying relatively smaller nucleic acid sequences.